Detection of negatively charged polymers using water-soluble, cationic, polythiophene derivatives

ABSTRACT

Novel methods allowing for the simple optical and electrochemical detection of double-stranded oligonucleotides are disclosed. The methods are rapid, selective and versatile. Advantageously, they do not require any chemical reaction on the probes or on the analytes since they are based on different electrostatic interactions between cationic poly (3-alkoxy-4-methylthiophene) derivatives and single-stranded or double-stranded (hibridized) oligonucleotides.

FIELD OF THE INVENTION

[0001] The present invention relates to simple and reliable methods fornegatively charged polymer detection, namely for sequence-selectivenucleic acid detection. More specifically, the present invention relatesto sequence-selective nucleic acid detection methods, which areessential for the rapid diagnosis of infections and a variety ofdiseases.

BACKGROUND OF THE INVENTION

[0002] Complexes of polythiophene derivatives bearing sulfonic acidmoieties and one or several adequately designed amine-containingmolecules (electrostatic interactions) have been shown to be responsiveto external stimuli (PCT/CA98/01082). More specifically, they were shownto undergo striking conformational changes when exposed to heat, lightor various chemical and biochemical moieties giving rise tothermochromism, photochromism, ionochromism or even biochromism. Thesesulfonic acid-bearing polythiophene derivatives are not positivelycharged and thus do not have any particular affinity for negativelycharged polymers.

[0003] The search for methods for sequence-selective nucleic aciddetection has evolved into an important research field and hassubsequently drawn the attention of researchers from various disciplinessuch as chemistry, physics, biochemistry, etc. As a result, someinteresting DNA hybridization sensors have recently been proposed.^(1,5)

[0004] However, most of these newly developed approaches performdetection by attaching a fluorescent or electro-active tag to theanalyte.

[0005] Assays that do not require nucleic acid functionalization priorto detection are of greater fidelity and several research groups havereported the utilization of conjugated field-responsive polymers(polypyrroles, polythiophenes, etc.) as electrochemical or opticaltransducers.^(6,7,23,24) Indeed, the ability of someoligonucleotide-functionalized conjugated polymers to transducehybridization events into an electrical or optical signal, withoututilizing any labeling of the analyte, has been demonstrated.⁸⁻¹⁰ Thedetection mechanism is based on a modification of the electrical and/oroptical properties through the capture of the complementaryoligonucleotides.

[0006] There thus remains a need for simpler, more sensitive and morereliable methods for the rapid and specific identification of nucleicacids. These nucleic acids could be used for the diagnosis of infectionsand disease. Ideally, an assay that does not require nucleic acidfunctionalization (chemical manipulation of nucleic acids) prior todetection nor complex reaction mixtures would have the followingcharacteristics: it would be simpler to use than the assays currentlyavailable and it would have a high degree of fidelity. Such an assaywould be highly beneficial and therefore very desirable.

[0007] The present invention seeks to meet these and other needs.

SUMMARY OF THE INVENTION

[0008] In general terms, the present invention relates to novelcationic, water-soluble polythiophene derivatives, which can readilytransduce oligonucleotide hybridization into a clearly interpretableoptical (colorimetric, fluorescent or luminescent) or electrical signal.These polymers can discriminate between specific and non-specifichybridization of nucleic acids differing by only a single nucleotide.

[0009] Specifically, the present invention relates to the synthesis anduse of cationic water-soluble polymers composed of thiophene monomershaving the following general formula:

[0010] wherein “m” is an integer ranging from 2 to 3; R* is a quaternaryammonium; Y is an oxygen atom or a methylene; and R¹ is a methyl groupor a hydrogen atom.

[0011] The present invention further comprises a method for detectingthe presence of negatively charged polymers, comprising the steps of:

[0012] a) contacting a complementary target to the negatively-chargedpolymer with a thiophene polymer to form a duplex;

[0013] b) contacting the duplex with the negatively charged polymer; and

[0014] c) detecting a change in electronic charge, fluorescence or coloras an indication of the presence of the negatively charged polymer.

[0015] The negatively charged polymers for the method as describedabove, may be selected from the group consisting of: acidic proteins,glycosaminoglycans, hyaluronans, heparin, chromatographic substrates,culture substrates and nucleic acids.

[0016] The present invention additionally comprises a method fordiscriminating a first nucleic acid from a second nucleic acid, thesecond nucleic acid differing from the first nucleic acid by at leastone nucleotide, comprising the steps of:

[0017] a) contacting a complementary target to the first nucleic acidwith a thiophene polymer to form a duplex;

[0018] b) contacting the duplex with the first nucleic acid, resultingin specific hybridization;

[0019] c) contacting the duplex with the second nucleic acid, resultingin non-specific hybridization; and

[0020] d) detecting a change in electronic charge, fluorescence or color

[0021] Finally, the present invention contemplates a number of specificapplications, such as the use of a positively charged polymer comprisinga repeating thiophene moiety for detecting the presence of negativelycharged polymers, and for purifying negatively charged polymers.

[0022] Further scope and applicability will become apparent from thedetailed description given hereinafter. It should be understood however,that this detailed description, while indicating preferred embodimentsof the invention, is given by way of illustration only, since variouschanges and modifications within the spirit and scope of the inventionwill become apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M(on a monomeric unit basis) solution of: a) polymer 2; b) polymer 2/X1duplex; c) polymer 2/X1/Y1 triplex, at 55° C. in 0.1M NaCl/H₂O using anoptical path length of 1 cm.

[0024]FIG. 2 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M(on a monomeric unit basis) solution of: a) polymer 2; b) polymer 2/X1duplex; c) polymer 2/X1/X1 mixture, at 55° C. in 0.1M NaCl/H₂O.

[0025]FIG. 3 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M(on a monomeric unit basis) solution of: a) polymer 2; b) polymer2/X1duplex; c) polymer 2/X1/Y2 mixture, at 55° C. in 0.1M NaCl/H₂O.

[0026]FIG. 4 shows the specific optical detection of Candida albicansversus Candida dubliniensis amplicons differing by only 2 nucleotides.

[0027]FIG. 5 shows a photograph of a polymer 2-oligonucleotide duplex(solution in the left tube was colored purple-red); a polymer2-hybridized oligonucleotides triplex (solution in the middle tube wascolored yellow); and a polymer 2 with partially hybridizedoligonucleotides with two mismatches (solution in the right tube wascolored pink).

[0028]FIG. 6 shows the formation of a duplex and a triplex betweenpolymer 2 and oligonucleotides.

[0029]FIG. 7 shows the fluorescence intensity during specifichybridization.

[0030]FIG. 8 shows the fluorescence intensity during non hybridization.

[0031]FIG. 9 shows the different steps involved in the electrochemicaldetection of DNA hybridization.

[0032]FIG. 10 shows the Cyclic voltammogram of polymer 1 onto an ITOmodified electrode (S=50 mm²) in the case of perfect hybridization(straight line) and in the case of a control blank (dashed line) in 0.1M NaCl/H₂O. Scan rate 100 mV/s (two successive cycles).

[0033]FIG. 11 shows the Cyclic voltammogram of polymer 2 onto an ITOmodified electrode (S=50 mm²) in the case of perfect hybridization(straight line) and in the case of a control blank (dashed line) in 0.1M NaCl/H₂O. Scan rate 100 mV/s (two successive cycles).

[0034]FIG. 12 shows the covalent attachment of a DNA probe onto aconducting surface, its subsequent hybridization with a complementaryDNA strand and its revelation with a conducting polymer.

[0035]FIG. 13 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M(on a monomeric unit basis) solution of a) polymer 2, b) polymer 2/X1duplex, c) polymer 2/X1/Y1 triplex, d) polymer 2/X1/Y2 mixture, and e)polymer 2/X1/Y3 mixture, at 55° C., in 0.1 M NaCl/H₂O. Capture probe X1:5′CATGATTGMCCATCCACCA 3′ and its perfect complementary target Y1: 3′GTACTMCTTGGTAGGTGGT 5′ are a DNA oligonucleotide pair specific forCandida albicans; capture probe X2: 5′ CATGATTGMGCTTCCACCA 3′ and itsperfect complementary target Y2: 3′ GTACTAACTTCGAGGTGGT 5′ are a DNAoligonucleotide pair specific for Candida dubliniensis; Y3: 3′GTACTMCTTCGTAGGTGGT 5′ is a complementary target DNA oligonucleotidedesigned to have one mismatch with both the C. albicans and the C.dubliniensis capture probes.

[0036]FIG. 14 shows a cyclic voltammogram of polymer 2 (SH-carbon₂₄-DNAwith polymer 2) bound to an ITO modified electrode (50 mm²) in the caseof perfect hybridization (X1/Y1; full line) and in the case of twocontrol blanks (X1/X1, dotted line and X1, dashed line) in 0.1 MNaCl/H₂O using a scan rate of 100 mV/s.

[0037]FIG. 15 shows a cyclic voltammogram of polymer 2 (SH-carbon₂₄-DNAwith polymer 2) bound to a gold modified electrode (50 mm²) in the caseof perfect hybridization (X1/N1; full line) and in the case of a controlblank (X1, dashed line) in 0.1 M NaCl/H₂O. The electrodes were submittedto a −0.4 V potential for 10 minutes before scanning; the scan rate usedwas 50 mV/s.

[0038]FIG. 16 shows a cyclic voltammogram of polymer 3 (SH-carbon₂₄-DNAwith polymer 3) bound to an ITO modified electrode (50 mm²) in the caseof perfect hybridization (X1/N1; full line) and in the case of twocontrol blanks (X1, dashed line and silanized ITO (no DNA), dash-dottedline) in 0.1 M NaCl/H₂O using a scan rate of 50 mV/s.

[0039]FIG. 17 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ M(on a monomeric unit basis) solution of: a) polymer 1/X1 duplex; b)polymer 1/X1/Y1 triplex, at 55° C. in 0.1 M NaCl/H₂O.

[0040]FIG. 18 shows the UV-visible absorption spectrum of a 2.4×10⁻⁵ (ona monomeric unit basis) solution of: a) polymer 4; b) X1/Y1/polymer 4triplex; c) X1/X1/polymer 4 mixture, at 25° C. in 0.1M NaCl/H₂O.

[0041]FIG. 19 shows the circular dichroism of a 1.2×10⁻⁴ M (on amonomeric unit basis) solution of polymer 3/X1/Y1 triplex at 55° C. in10 mM Tris buffered solution containing 0.1M NaCl.

[0042]FIG. 20 shows the UV-visible spectroscopy spectrum of 7.9×10⁵ Msolutions in 0.1M NaCl and TE, at 55° C. of a) polymer 2; b) duplex(X1+polymer 2); c) triplex (X1+Y1+polymer 2); d) triplex with 2mismatches (X1+5′ TGGTGGATGCATCAATCATG 3′), triplex with 3 mismatches(X1+5′ TGGTGGATACATCAATCATG 3′), triplex with 5 mismatches (X1+5′TGGTGGAAACAACMTCATG 3′); e) triplex with 1 mismatch (X1+5′TGGTGGATGCTTCAATCATG 3′).

[0043]FIG. 21 shows the UV-visible spectroscopy spectrum of 7.9×10⁻⁵ Msolutions in 0.1M NaCl and TE, at 55° C. of a) polymer 2; b) duplex(X1+polymer 2); c) triplex (X1+Y1+polymer 2); d) triplex with 2mismatches (X1+Y2), (X1+5′ TGGTAGATGCTTCAATCATG 3′), (X1+5′TGGTGGTTGCTTCAATCATG 3′), (X1+5′ TGGTGGATGCTTTTAATCATG 3′), (X1+5′TGGTGGATGCTTCATTCATG 3′), (X1+5′ TGGTGGATGCTTCAATTATG 3′).

DETAILED DESCRIPTION OF THE INVENTION

[0044] Other objects and attendant features of the present inventionwill become readily appreciated, as the same becomes better understoodby reference to the following detailed description of the inventiondescribed for the purpose of illustration.

[0045] In a broad sense, the invention provides novel cationic, watersoluble polythiophene derivatives that produce a clearly interpretableoptical (colorimetric, fluorescent or luminescent) or electrical signal,when bound to negatively charged polymers.

[0046] The present invention provides for polymers capable ofdiscriminating between specific and non-specific hybridization ofnucleic acids differing by only a single nucleotide.

[0047] The present invention provides improved research tools. Morespecifically, a means for detecting nucleic acids from eucaryoticorganisms as well as prokaryotic organisms such as Bacteria and Archaea.

[0048] The present invention also provides for the development of newnucleic acid detection technology, and more specifically, new detectiondevices based on the use of the polythiophene derivatives of the presentinvention.

[0049] The present invention further provides for improved clinicaldiagnostics, that is, the detection of infectious agents, the diagnosisof genetic diseases and tools useful for use in the pharmacogenomicsfield.

[0050] The present invention further provides for improved medico-legal(forensic) diagnostics, more specifically the filiation of people andanimals, “forensic” tools and other genetic testing tools.

[0051] The present invention also provides for improved plantidentification.

[0052] The present invention also provides for environmental andindustrial screening, more specifically for the detection of geneticallymodified organisms, the detection of pathogenic agents, alimentarytracability, the identification of organisms of industrial interest(e.q. alimentary, pharmaceutical or chemical fermentation and soildecontamination).

[0053] The present invention also provides for polythiophenes having anaffinity for negatively charged polymers such as nucleic acids andglycosaminoglycans of natural or synthetic origin, allowing for thepurification of these polymers. For example, when a polythiophene iscoupled to a solid support, nucleic acids can be purified by affinityand/or ion exchange chromatography.

[0054] The present invention also provides for polythiophenes that arethermostable and autoclavable, allowing for a wide range ofapplications.

[0055] The present invention further provides methods and tools by whichnegatively charged polymers such as acidic proteins (kinesins),glycosaminoglycans (hyaluronans, heparin) and any natural or syntheticnegative polymers can be detected or blocked by binding with thepolythiophenes.

[0056] Advantageously, this novel approach is rapid, specific,sensitive, and highly versatile, yet simple. It is based on thedifferent electrostatic interactions and conformational structuralchanges between single-stranded or double-stranded negatively-chargedoligonucleotides or nucleic acid fragments, and cationic electroactiveand photoactive poly(3-alkoxy-4-methylthiophene) derivatives. It allowsa single reagent assay procedure for genomic analysis and moleculardiagnostics. Furthermore, the above mentioned detection methods can alsobe used for solutions of nucleic acids, nucleic acids separated by gelelectrophoreses, nucleic acids fixed onto solid supports such as glassslides or plates, silicon chips or other polymers.

[0057] Synthesis

[0058] Set forth below are preferred synthesis schemes for thepreparation of the water-soluble, cationic, electroactive andphotoactive poly(3-alkoxy-4-methylthiophene)s.

[0059] The synthesis of monomer 1 is carried out in a two-stepprocedure, starting from 3-bromo-4-methylthiophene (Aldrich Co.) (seeScheme 1). The first step is a nucleoplilic substitution reaction ontothe thiophene ring catalyzed by Cul, as reported by El Kassmi et al.¹¹The second step involves a quaternization reaction between the tertiaryamine and 1-bromoethane in acetonitrile.¹²

[0060] Similarly, monomer 2 is prepared from3-(2-bromoethoxy)-4-methylthiophene (compound 4) and 1-methylimidazole(Aldrich Co.) (see Scheme 2). Compound 4 is prepared according to theprocedure developed by Leclerc et al.¹³ The quaternization reactionbetween 1-methylimidazole and compound 4, provides the desired monomerimidazolium salt 2.¹⁴

[0061] Similarly, the synthesis of monomer 3 involves the conversion of3-thiopheneethanol (compound 5) into its corresponding mesylateprotected derivative (compound 6). The quaternization reaction between1-methylimidazole and compound 6, provides the desired monomerimidazolium salt 3.

[0062] The synthesis of monomer imidazolium salt 4 involves aquaternization reaction of compound 4 with 1,2-dimethylimidazole.

[0063] A more general procedure reflecting the preparation of cationicthiophene monomers is depicted in scheme 5, wherein “m” is an integerequal to 2 or 3; “Y” is an oxygen atom or a methylene group; and R¹ is ahydrogen atom or a methyl group.

[0064] When R* is Et₃N, Y is an oxygen atom and R¹ is a methyl group,then “m” is equal to 3. When R* is 1-methylimidazole, Y is an oxygenatom and R¹ is a methyl group, then “m” is equal to 2. When R* is1-methylimidazole, Y is a methylene group and R¹ is a hydrogen atom,then “m” is equal to 2. When R* is 1,2-dimethylimidazole, Y is an oxygenatom and R¹ is a methyl group, then “m” is equal to 2.

[0065] The inherent chemical and physical properties of imidazoleprovide for a wide electrochemical window, favorable for electrochemicaldetection.¹⁵

[0066] All polymers, more specifically the cationic, water soluble,electroactive polymers 1, 2, 3 and 4 (Scheme 6), were synthesized byoxidative chemical polymerization of the corresponding monomers usingFeCl₃ or K₂S₂O₈ as the oxidizing agent in chloroform. This method ofpolymerization yields well-defined regio-regular3-alkoxy-4-methylthiophene polymers (1, 2 and 4) as well as anon-regioregular 3-alkylthiophene polymer (3), having an averagemolecular weight of about 5 kDa and a polydispersity index of ca. 3.¹⁶Note that “n” can vary from 3 to about 100. The resulting polymers(using FeCl₃ as the oxidizing agent) contain a mixture of anions such asFeCl₄ ⁻Cl⁻and Br⁻. In order to produce a cationic polymer with only onespecific counter anion (e.g. hydrophilic counter anions like Cl⁻, Br⁻,I⁻, CH₃SO₃ ⁻, etc. or hydrophobic counter anions like BF₄ ⁻, CF₃SO₃ ⁻,PF₆ ⁻, etc.), an anionic-exchange reaction is performed by dialysis orprecipitation. As expected, all resulting polymers were found to besoluble in aqueous solutions when in the presence of hydrophilic anions.

[0067] As any water-soluble cationic polyelectrolytes, the polythiophenederivatives of the present invention can make strong complexes withnegatively-charged oligomers and polymers.¹⁷ This complexation resultsin the formation of complexes having specific optical properties. Forinstance, at 55° C., aqueous solutions (0.1 M NaCl or 10 mM Trisbuffer/0.1M NaCl) of the cationic polymer 2 are yellow (λmax=397 nm).This absorption maximum at a relatively short wavelength is related to arandom coil conformation of the polythiophene derivative.⁶ After theaddition of one equivalent (on a monomeric unit basis) of a givenoligonucleotide (20-mers), the mixture becomes red (λ_(max)=527 nm) dueto the formation of a so-called duplex. After 5 minutes of mixing in thepresence of one equivalent of a complementary oligonucleotide, thesolution becomes yellow (λ_(max)=421 nm) presumably due to the formationof a new complex (triplex) (FIGS. 1 and 5).

[0068] A schematic description of these conformational transitions forboth types of polyelectrolytes is given in FIG. 6.

[0069] Based on previous studies on thermochromic, solvatochromic andaffinitychromic regioregular poly(3-alkoxy-4-methylthiophene)s,⁶ it isbelieved that these calorimetric effects are made possible due to thedifferent conformational structure of the conjugated polymer in theduplex (highly conjugated, planar conformation) compared to thatobserved in the triplex (less conjugated, non-planar conformation) andto a stronger affinity of the conjugated polymer for the double-strandedoligonucleotides (nucleic acids) (1×10⁵ M⁻¹) than that measured forsingle stranded oligonucleotides (nucleic acids) (5×10⁴ M⁻¹).

[0070] In a control experiment it was demonstrated that the addition tothe solution of an oligonucleotide identical to that of the captureprobe results in no color change (FIG. 2).

[0071] In order to verify the specificity of these complexations, twopairs of complementary oligonucleotides (20-mers) differing by only 1 or2 nucleotides were synthesized (Table 1) and carefully investigated. Aslight but distinct change in the UV-visible absorption spectrum isobserved in the case of the oligonucleotide target having 2 mismatches.Even with only one mismatch, it is possible to distinguish between aperfect and a non-perfect hybridization (FIG. 13). In this case, thecalorimetric difference is mainly based on different kinetics ofcomplexation, since similar yellow aqueous solutions are observed after30-60 minutes of mixing at 55° C. However, it is possible to stop thehybridization reaction after 5 minutes of mixing, by placing thesolutions at room temperature. Following these procedures, stable yellowand orange solutions are obtained (curves c and e) (FIG. 13). Thedetection limit of this calorimetric method is about 1×10¹³ molecules ofoligonucleotide (20-mers) in a total volume of 100 μL.

[0072] Very similar results have been obtained for polymer 1 (FIG. 17)and polymer 4 (FIG. 18) and various oligonucleotides. FIG. 20 shows theUV-Visible absorbance spectrum of polymer 2 when using targetoligonucleotides ranging from 0 to 5 mismatches. FIG. 21 illustrates theUV absorbance results of polymer 2 using the target oligonucleotidealways having two mismatches at different positions. These results showthat the polymer can discriminate between perfectly matched andmismatched hybrids, independently of the nature of the mismatchednucleotide bases, and independently of the position or the length of themismatches. Moreover, it is even possible to discriminate a singlemismatch from multiple mismatches. TABLE 1 Synthetic oligonucleotides tostudy the specificity of hybridization. Oligonucleotides Oligonucleotidespecific specific for Candida albicans for Candida dubliniensis X1 X2 5′5′ CATGATTGAACCATCCACCA CATGATTGAAGCTTCCACCA 3′ 3′ Y1 Y2 3′ 3′GTACTAACTTGGTAGGTGGT GTACTAACTTCGAAGGTGGT 5′ 5′ Y3 (designed to have onemismatch wit X1 and X2) 3′ GTACTAACTTCGTAGGTGGT 5′

[0073] The concentration of a particular sequence region of DNA can beamplified by using a polymerase chain reaction (PCR), and the presentcolorimetric method can be extended to these PCR products. Indeed, theintroduction of the polymerase chain reaction (PCR) has solved theproblem of detecting small amounts of DNA and the polymers of thepresent invention can be used in the identification of PCR products. UVspectroscopic results in the absorbance range of 430-530 nm (FIG. 4)have illustrated the specific optical detection of Candida albicans andCandida dubliniensis amplicons, which differ by only 2 nucleotides, by apolymer 2/X1 duplex. Such detection was carried out in 45 minutesdirectly from a PCR product at a concentration normally generated in a100 μL PCR volume (ca. 3×10¹² copies). Experimental details on PCR andchoice of target sequences for identification of Candida are provided ina co-pending patent application (PCT/CA00/01150).

[0074] In addition, as shown in FIG. 19, circular dichroism (CD)measurements reveal an optical activity for polymer 3 in its randomcoil, a bisignate CD spectrum centered at 420 nm in the triplex,characteristic of a right-handed helical orientation of thepolythiophene backbone. Such a right-handed helical structure iscompatible with binding of the polymer to the negatively-chargedphosphate backbone of DNA. The thermal stability study by UV or CDmeasurements shows a different thermal stability between a duplex and atriplex, and this property could be extremely useful for more stringentwashing conditions.

[0075] A fluorometric detection of oligonucleotide hybridization is alsopossible based on the difference in the fluorescence quantum yield ofthe positively-charged poly(3-alkoxy-4-methylthiophene) in the randomcoil (the isolated state) or in the aggregated state.¹⁶ For instance, at55° C., the yellow appearance of polymer 2 is fluorescent (quantum yieldof 0.03) but upon addition of one equivalent of a negatively-chargedoligonucleotide, the intensity of the emission spectrum is stronglydecreased (quenched). In the case of perfect hybridization, thepolymeric triplex gives a stronger emission (FIG. 7). Upon addition ofthe same oligonucleotide, no hybridization occurs and the solution doesnot show any change in fluorescent intensity (FIG. 8). Using a laser asthe excitation source, very low limits of detection are obtained. It ispossible to detect the presence of as few as 3×10⁶ molecules of thecomplementary oligonucleotide (20-mers) in a volume of 200 μL, whichcorresponds to a concentration of 2×10⁻¹⁴ M. Moreover, by covalentlyattaching the oligonucleotide to a fluorescent-conjugated polymer, or byusing an optimized fluorescence detection scheme based on a highintensity blue diode (excitation source) and a non-dispersive,interference filter-based system, an even more sensitive and morespecific detection capability is achieved.

[0076] The electrochemical properties of polymers 1 and 2 can be usedfor the detection of DNA hybridization in aqueous solutions, as shown inFIG. 9.^(8,10) Using the layer by layer deposition techniques,^(25.26)the first step required the binding of a capture probe composed ofsingle-stranded oligonucleotide X1 to an ammonium-functionalized indiumtin oxide (ITO) surface.¹⁸²⁷ After rinsing with pure water, the modifiedelectrode so-obtained was dipped and hybridized in the presence of acomplementary oligonucleotide Y1. The resulting electrode was revealedwith an aqueous solution of positively-charged polymer 1 or 2 (10⁻⁴ M ona monomeric basis), which provides a signal that is a function of theamount of DNA present on the surface. As a control experiment, anaqueous solution of oligonucleotide X1 was added to the X1 modified ITOelectrode, and then transferred into an aqueous solution of polymer 1 or2. In this way, these polymers serve as “mass transducers” for theoligonucleotide present in the sample.

[0077] The detection of DNA hybridization is further demonstrated by thefollowing two examples using different cationicpoly(3-alkoxy-4-methylthiophene)s (polymers 1 and 2). The so-obtainedresults are illustrated in FIGS. 10 and 11.

[0078] In both cases, the maximum anodic current is more important inthe case of perfect hybridization as compared to the blank control. Inaddition, a shift to a higher potential (ca. 40-50 mV) is observed whenspecific hybridization occurs (38 mV for polymer 4 as compared to 52 mVfor polymer 2). The higher oxidation current can be explained by thestronger affinity of the polymers for double-stranded oligonucleotides,whereas the positive shift of oxidation potential is explained by theformation of a less conjugated structure in the case of specifichybridization. This is in agreement with previous optical measurements.

[0079] An assay using a smaller electrode [S(surface)=10 mm²] hasallowed the detection of 2×10¹¹ molecules of oligonucleotide (20-mers).This very simple electrochemical methodology is already more sensitive,by two orders of magnitude, than the best results obtained withelectrochemical methods using oligonucleotide-functionalized conjugatepolymers.¹⁰ Clearly, by decreasing the size of the electrodes and byincreasing the size of the target molecules, much lower detection limitsshould be obtained.

[0080] In order to further enhance the specificity of the detection, theoligonucleotide probe can be covalently attached to the polythiophenederivatives

[0081] The detection of DNA sequences can also be carried outelectrochemically as shown below in Scheme 7.

[0082] The DNA probes can be covalently fixed to a conductive surface(FIG. 12). This allows for the linking of a larger number of DNA probesto the surface, improving the specificity and detection limit for asmall surface.

[0083] The first step involves the modification of the conductivesubstrate (ITO, SnO₂, gold, doped silicon or other conductive substrate)by the covalent attachment of a single-stranded DNA probe. Thecomplementary DNA strand is hybridized and the polymer is captured onthe hybridized probe. The observed electrochemical signal is differentfor a hybridized probe (ds-DNA) versus a non-hybridized probe (ss-DNA).A linker can be used to attach the DNA probe to the conductive surface.The end groups of the linker are such that one end readily reacts withthe conductive substrate to form a covalent bond, whereas the other endreadily reacts with an “end-modified” (SH, NH₂, COOH, etc.) DNA probe,with or without a spacer (carbon₂₄) between the reactive function andthe DNA.^(19,20,21) The end group of the linker that reacts with theconductive substrate's surface can be a silane derivative, such as analkoxysilane, or a chlorosilane. The other end group of the linker canbe composed of an aldehyde, a carboxylic acid, a primary amine, asuccinimide ester moiety or other functional groups capable of reactingwith “end-modified” DNA probes, that is, DNA probes having specific endgroups, resulting in the formation of covalent bonds with or without thehelp of coupling agents.

[0084] The immobilization of an oligonucleotide by means of a thiolgroup can be carried out through its 3′ or 5′ terminal group.Accordingly, DNA was immobilized onto an ITO surface following theliterature procedure for immobilizing DNA on a glass surface.^(28,29,30)

[0085] It is also possible to link “end-modified” DNA probes to variousother surfaces such as gold or other metals and metal oxides.²²Non-metallic surfaces such as beads, glass slides, optical fibers or anyother suitable non-metallic solid support, are also possible. Theimmobilization of DNA onto a gold surface is accomplished followingknown published techniques, and the density of the attached probes canvary depending on the concentration and reaction times.^(31,32) Thehybridization efficiency can be optimized by heating the substratebefore adding the complementary strand.^(31,33) Polymer deposition canbe optimized by dipping the substrate vertically into a polymersolution, and by varying the salt concentration and the temperature ofthe polymer solution. The washes can be further optimized to limit thecontribution of the polymer with the non-hybridized probe.

[0086] The signal observed for a triplex should be stronger than thatobserved for a duplex, with possibly a small shift towards higherpotentials when hybridization occurs. The amount of DNA is higher in thecase of a triplex, which implies the presence of a higher amount ofnegative charges. It is suspected that there might be two equivalents ofpolymer (positive charge) binding in the case of a triplex. Moreover, inthe case where only one equivalent of polymer is bound to DNA, thepolymer on a non-hybridized probe (ss-DNA) is more easily washed off, ascompared to a polymer bound to DNA on a hybridized probe (ds-DNA), sinceit is less stable at high salt content or elevated temperatures. Bywashing off essentially all of the polymer from the duplex while leavingthe triplex intact, a signal coming directly from the triplex(hybridized probe+polymer) is assured. Finally, targets havingmismatches will give a different signal and possibly a lower current,due to less perfect hybridization.

[0087] The electrochemical detection of DNA hybridization is furtherdemonstrated by using polymer 2 and polymer 3, as illustrated in FIGS.14-16.

EXPERIMENTAL SECTION Example 1 General Procedure for the AromaticNucleophilic Substitution on Thiophene; Synthesis of Compound 3

[0088] Sodium hydride (0.4 g, 15 mmol) was added between 0 and 10° C.under nitrogen to a solution of N,N-diethylpropanolamine (2.0 g; 15.2mmol) in 50 mL of DME, and the resulting mixture stirred at ambienttemperature for 20 minutes. 3-Bromo-4-methyl thiophene (2.0 g, 11.3mmol) dissolved in 20 mL of DME (20 mL) and CuI (1.07 g, 5.65 mmol) werethen added to the reaction mixture. The mixture was subsequently stirredovernight at 95° C., while under nitrogen, diluted with methylenechloride and filtered. The organic phase was washed three times withwater, dried over MgSO₄ and evaporated. The crude product was purifiedby chromatography on silica gel using CH₂Cl₂ as the eluent, followed bythe use of MeOH. Compound 3: Yield 26%; ¹H NMR (CDCl₃) δ: 1.04 (t, 6H);1.93 (m, 2H); 2.09 (s, 3H); 2.57 (m, 6H), 3.97 (t, 2H); 6.14 (d, 1H);6.81 (d, 1H); ¹³C NMR (CDCl₃) δ: 11.69; 12.50; 26.84; 46.91; 49.26;68.19; 95.88; 119.57; 129.05; 156.04; MS m/e: Calcd. For C₁₂H₂₁N₀O₁S₁;227.1344; Found: 227.1347.

Example 2 General Procedure for Quaternization Reaction: Synthesis ofMonomer 1

[0089] 1-Bromoethane (6 mL, 80.4 mmol) was added to a solution ofcompound 3 (0.49, 1.8 mmol) in 60 mL of acetonitrile. The reactionmixture was stirred at 70° C. under nitrogen for 3 days. Afterevaporation of the acetonitrile, the crude product was crystallized fromethyl acetate as a colorless powder.

[0090] Monomer 1: Yield 100%; m.p. 145-147° C.; ¹H NMR (CDCl₃) δ: 1.41(t, 9H); 2.06 (s, 3H); 2.31 (m, 2H); 3.57 (m, 8H), 4.12 (t, 2H); 6.24(d, 1H); 6.84 (d, 1H); ¹³C NMR (CDCl₃) δ: 7.87; 12.61; 22.55; 53.61;54.66; 65.80; 97.18; 120.28; 128.26; 154.77.

Example 3 Synthesis of Monomer 2

[0091] 1-Methyl-imidazole (1.0 mL, 12.3 mmol) was added to a solution ofproduct 4 (0.54 g, 2.46 mmol) dissolved in CH₃CN (35 mL). The resultingreaction mixture was stirred at 70° C. for two days. After evaporationof the solvent, the crude product was washed twice with warm ethylacetate and twice with diethyl ether at room temperature to providemonomer 2 as a pure white solid compound.

[0092] Monomer 2: Yield 88%;. 92-94° C.; ¹H NMR (CDCl₃) δ: 2.05 (s, 3H);4.09 (s, 3H); 4.38 (t, 2H); 4.90 (t, 2H), 6.27 (d, 1H); 6.82 (d, 1H);7.63 (s, 1H); 7.68 (s, 1H); 10.24 (s, 1H); ¹³C NMR (CDCl₃) δ: 12.81;36.76; 49.36; 68.01; 97.92; 120.68; 123.26; 123.30; 128.35; 137.51;154.23.

Example 4 Synthesis of Monomer 3

[0093] Methanesulfonyl chloride (2.4 mL; 31.2 mmol) was added dropwiseto a solution of 3-thiopheneethanol (2.0 g; 15.6 mmol) and triethylamine(4.3 mL; 31.2 mmol), in dichloromethane (50 mL). The reaction mixturewas stirred at room-temperature for two hours. The organic phase waswashed with a NaHCO₃ solution, followed by several washings with water,and was finally dried with MgSO₄ and concentrated. The crude product waspurified by silica gel chromatography using CH₂Cl₂/hexane (1/1) as theeluent to yield compound 6 (59%); ¹H NMR (300 MHz, CDCl₃, 25° C., TMS):δ=2.87 (s, 3H); 3.08 (t, 2H); 4.40 (t, 2H); 6.98 (d, 1H); 7.09 (d, 1H);7.29 (dd, 1H). ¹³C NMR (300 MHz, CDCl₃, 25° C., TMS): δ=29.99; 37.19;69.59; 122.24; 125.98; 127.98; 136.44.

[0094] 1-Methyl imidazole (0.4 g; 4.85 mmol) was added to a solution ofcompound 6 (0.2 g; 0.97 mmol) in toluene (20 mL). The reaction mixturewas stirred at 94° C. for two days. Following evaporation of thesolvent, the crude product was washed with warm ethyl acetate to yieldmonomer 3 as a liquid.

[0095] Monomer 3: Yield (95%); ¹H NMR (300 MHz, CDCl₃, 25° C., TMS):δ=2.72 (s, 3H); 3.19 (t, 2H); 3.90 (s, 3H); 4.49 (t, 2H); 6.95 (d, 1H);7.06 (d, 1H); 7.24 (m, 2H); 7.33 (s, 1H); 9.68 (s, 1H). ¹³C NMR (300MHz, CDCl₃, 25° C., TMS): δ=30.77; 36.10; 39.67; 50.06; 122.24; 122.81;123.01; 126.46; 127.66; 136.08; 137.80.

Example 5 Synthesis of Monomer 4

[0096] The quaternization reactions were carried out following theprocedure described in example 3.

[0097] Monomer 4: Yield 85%; ¹H NMR (300 MHz, CDCl₃, 25° C., TMS):δ=2.02 (s, 3H); 2.89 (s, 3H); 3.96 (s, 3H); 4.39 (t, 2H); 4.85 (t, 2H);6.27 (d, 1H); 6.83 (d, 1H); 7.57 (d, 1H); 7.96 (d, 1H).

Example 6 General Procedure for the Chemical Polymerization: Synthesisof Polymer 1

[0098] To a solution of iron trichloride (0.94 g, 5.8 mmol) inchloroform (23 mL) under nitrogen, a solution of monomer 1 (0.487 g, 1.4mmol) in chloroform (15 mL) was added dropwise. The mixture was stirredat room temperature for a period of 2 days. The reaction mixture wasevaporated to dryness and the crude product washed quickly withmethanol, and dissolved in excess of acetone, and precipitated by theaddition of an excess of tetrabutylammonium chloride ortetrabutylammonium bromide. The black-red polymer was dissolved inmethanol and dedoped by adding a few drops of hydrazine. The finalsolution was evaporated. The resulting polymer was washed several timeswith a saturated solution of tetrabutylammonium chloride ortetrabutylammonium bromide in acetone, and by Soxlet extraction withacetone over a period of 6 hours, and then dried under reduced pressureto yield polymer 1 (0.32 g, 66%).

Example 7 General Procedure for Optical Detection

[0099] i) Synthetic Oligonucleotides

[0100] In a quartz UV cuvette, 100 μL (7.47×10⁻⁸ repeat units (RU) ofpositive charges) of a solution of polymer 2 were added to an aqueoussolution (3 mL) containing either 0.1M NaCl or 10 Mm Tris buffer plus0.1M NaCl (pH=8). The mixture was heated at 55° C. for 5 min and had ayellow appearance. 12 μL (7.47×10⁻⁸ RU of negative charges) ofoligonucleotide solution (capture probe) were then added and theresulting red solution kept at 55° C. for an additional 5 minutes. Theappropriate oligonucleotide target was added to the solution at 55° C.over 5 minutes. A final yellow color is indicative of a positive result,meaning that perfect hybridization has taken place. On the other hand, ared or a red-pink color is representative of non specific or partialhybridization (two mismatches), respectively (FIGS. 1-3, respectively).

[0101] ii) PCR Products

[0102] The amplicon (double stranded 149 base pairs) from the PCRproduct was pre-purified by <<QlAquick>> purchased from Qiagen. H₂O (90μL) was added to a centrifuge tube followed by the addition of 6.7 μL(5.03×10⁻⁹ mol of positive charge) of a solution of polymer 2, followedby the addition of the oligonucleotide capture probe Y1 (2 μL; 5.03 10⁻⁹mol of negative charge) and finally, by the addition of NaCl 1M (20 μL).The resulting mixture was heated at 50° C. for 10 min. The purified PCRproduct was freshly denatured, cooled in ice water and then added to theabove solution. The hybridization reaction was kept at 50° C. for 35 minand the color change observed either visually or by UV measurement (FIG.4).

Example 8 General Procedure for Fluorescent Detection

[0103] A procedure identical to that described for optical detection wasemployed, with the exception of the use of a fluorospectrometer. Thefluorescent intensity of a “duplex” (association between a positivelycharged polymer and an oligonucleotide capture probe) was weak orinsignificant (practically zero) due to the fluorescent-quenchingproperty of the aggregated form of the polymer. When perfecthybridization does occur, the fluorescent signal becomes moresignificant (FIG. 7).

Example 9 General Procedure for Electrochemical Detection

[0104] The electrochemical test for hybridization was performed inconjunction with a control blank. 60 μL (1.44×10⁻⁸ mol of negativecharge) of captured oligonucleotide Y1 were deposited on the aminatedITO electrode (S=50 mm²) at ambient temperature for 5 min. After washingwith water, 60 μL (1.44×10⁻⁹ mol of negative charge) of the targetoligonucleotide X1 were added and the hybridization carried out at 55°C. over 20 minutes. The electrode was then cooled to room temperatureover 10 min and washed twice with 0.3 M NaCl, 0.03 M NaOAc and 0.1% SDS(pH 7) and water. 100 μL (1×10⁻⁸ mol of positive charge) of a solutionof polymer 1 or 2 was spread on the modified electrode for 5 min,followed by washing with CH₃CN/H₂O (1/4) and water. Cyclic voltammogramswere performed in aqueous 0.1 M NaCl solutions (FIGS. 10 and 11).

Example 10 General Procedure for the Preparation of ElectrodesSubstituted With Covalent Probes

[0105] i) Covalent Attachment of DNA Probes to an ITO Electrode (Polymer2)

[0106] ITO slides are sonicated in hexanes (10 min), methanol (10 min)and ultrapure water (10 min), and are then treated with an aquaregiasolution (H₂O₂/H₂O/NH₄OH, 1/5/1) at 40° C. over a 30 minute period. Theresulting slides are rapidly washed with water, followed by sonicationin water and in acetone, then dried with air, nitrogen or argon, andheated to 110° C. for 2 to 10 min. Following this, the slides aresubmerged for three hours, while under an inert atmosphere, in anacidified ethanol solution (1 mM acetic acid in 95% ethanol) containing5% mercaptopropyltrimethoxysilane, followed by sonication in freshethanol (95%) and in ultrapure sterile water. Finally, the slides areheated at 110° C. for at least one hour, and cooled to room temperaturebefore modification with DNA.

[0107] The washes, following DNA deposition, hybridization and polymerdeposition, are carried out using an orbital shaker, unless otherwisestated. DNA attachment is carried out using a solution ofSH-carbon₂₄-DNA in sodium citrate buffer (30 mM, pH=4, 50 μL) which isdeposited onto each ITO slide, each spot forming a circle of about 1 cmin diameter. The deposition reaction is performed in a humidifiedchamber over a period of 20-24 hours. Any non-deposited DNA is drainedoff, and the slides washed with 5×SSC+0.1% Tween 20, followed bywashings with 1×SSC+0.1% Tween 20 and NaCl 0.1M.

[0108] ii) Hybridization (ITO, Polymer 2)

[0109] 50 μL of RC-DNA (Y1) or of probe X1 (blank test) (25 μM in2×SSC), are deposited onto the DNA spot. The slides are then heated to55° C. for about 3 hours while in a humid atmosphere, and then cooled toroom temperature for 15 minutes. Any non-reacted DNA is drained off andthe slides washed with 2×SSC, NaCl (0.1M), with 1×SSC+0.1% Tween 20 andwith NaCl (0.1M).

[0110] iii) Polymer Deposition (ITO, Polymer 2)

[0111] Polymer 2 is deposited onto the electrodes. The slides are dippedvertically over a period of 5 minutes in a solution of polymer 2 (2 mLof 10⁻⁴ M in 0.01M NaCl) at room temperature. The slides are then dippedin 2 mL of a NaCl solution (0.01 M), followed by dipping in 2 mL of aCH₃CN/H₂O (1/4) solution and finally, by dipping in 2 mL of a NaClsolution (0.01M).

[0112] iv) Hybridization (ITO, Polymer 3)

[0113] 30 μL of Y1 (2.5 μM/NaCl 0.1M) are inserted into hybridizationchambers which are placed onto the surface of the ITO electrode. Theslides are heated for about two hours at 55° C. while in a humidatmosphere, and then cooled to room temperature. The hybridizationchambers are removed and the slides washed with a 0.1M NaCl solution andfinally dried with argon.

[0114] v) Polymer Deposition (ITO, Polymer 3)

[0115] Polymer 3 is deposited onto the electrodes. A 30 μL aqueoussolution of polymer 3 (10⁻⁴M) is inserted into hybridization chamberswhich are then placed onto the surface of the ITO electrode. The slidesare heated at 55° C. for 20 minutes. The hybridization chambers areremoved and the slides washed with a first portion of a 0.8M NaClsolution at 55° C., then washed with a new portion while cooling to roomtemperature. The slides are finally rinsed at room temperature with a0.1M NaCl solution.

[0116] vi) Covalent Attachment of DNA Probes to a Gold Electrode(Polymer 2)

[0117] Gold slides are rinsed sequentially with hexanes, methanol andwater. The plates are then treated with a pyranah solution (H₂O₂30%/H₂SO₄ concentrated; 30/70) over a period of about 15 minutes. Theslides are then thoroughly washed with nanopure sterile water, and driedwith argon.

[0118] DNA deposition is performed under inert atmosphere using 50 μL ofa solution of SH-carbon₂₄-DNA (25 μM in 1M phosphate buffer (pH=7);K₂HPO₄+KH₂PO₄). The solution is deposited onto a 1 cm² surface, andtreated for about 16 hours under inert atmosphere. Any non-reacted DNAis then drained off, and the slides sequentially washed with H₂O, 2×SSC,and sterile water.

[0119] Polymer 2 is deposited onto the electrodes. A 50 μL solution ofpolymer 2 (10⁻⁵ M in 0.1M NaCl) is deposited onto the DNA spot and theslides heated to 55° C. for about 30 minutes. The slides are then washedrepeatedly with a NaCl solution (0.1M) at room temperature.

[0120] Note that the order of additions does not have to follow thatdescribed in the above example, that is, a complementary (or anon-complementary or with mismatches) DNA strand is added to acovalently linked DNA strand, followed by the addition of a polymersolution. The polymer solution may be added prior to the addition of thecomplementary (or a non-complementary or with mismatches) DNA strand.

Example 11 The Use of the Polymers of the Present Invention to PurifyNucleic Acids and Other Such Negatively Charged Molecules

[0121] The polymers described in the present invention have highaffinity for negatively charged molecules, especially nucleic acids. Inaddition, they are soluble in aqueous solution and very stable under awide range of temperatures. Therefore, it is possible to use theseproperties to purify nucleic acids and other negatively chargedmolecules. Chromatographic separation would involve the following steps:(1) immobilizing the polymers; (2) applying the analyte to be separatedonto the immobilized polymers under conditions such that electrostaticinteractions are possible between the analyte and the polymer; and (3)eluting the analyte by applying conditions where electrostaticinteractions between the analyte and the polymer are not favored.

[0122] Immobilization of the polymer can be achieved by covalentlycoupling the polymer to a suitable solid support such as glass beads, orto beads made of other types of polymers. Alternatively, coupling to asolid support could be achieved via electrostatic interactions, such asthat demonstrated in Example 10. In the latter case, where a captureoligonucleotide is covalently attached to a solid support, the polymerwould be expected to be eluted along with the analyte in the finalelution step since it is attached only by electrostatic bonds. Colorchanges could even be used to monitor the chromatographic process.

[0123] Conditions for binding the analyte to the polymer wouldpreferably involve solutions of low ionic charge, such as water, 0.1 MNaCl and 10 mM Tris buffer/0.1 M NaCl. Different washing conditions canbe applied to remove any unwanted fractions of the analyte. pH changescould alternatively be used to obtain conditions for binding the analyteto the polymer.

[0124] Conditions for eluting the analyte involves solutions of highionic charge, such as 1.0 M NaCl solution or any other solutioncomprising a sufficiently high counter ion concentration capable ofcompeting for the electrostatic interactions with the polymer. Again, pHchanges could alternatively be used to obtain conditions for eluting theanalyte.

Example 12 Detection of Messenger RNA

[0125] Purified, in vitro transcribed, polyadenylated messenger RNA ofArabidopsis thaliana gene coding for NAC1, was purchased fromStratagene. Perfectly matched complementary DNA oligonucleotide N1 (5′CGAGGCTTCCATCAATCTTA 3′) was synthesized by phosphoramidite chemistry ona Perkin-Elmer 391 synthesizer. Unrelated Y2 DNA oligonucleotide wasobtained in the same manner. All reagents and liquid handling materialcoming into contact with the RNA were either certified RNAse-free ortreated with diethylpyrocarbonate (J. Sambrook and D. W. Russel,Molecular Cloning, A Laboratory Manual, CSHL Press, 2001) to inactivateRNAses. Fluorescence measurements were performed at 42.5° C. on aVariant Cary Eclipse spectrofluorometer with excitation set at 420±10 nmand fluorescence emission measured at 53015 nm, applying 1000 volts tothe detector.

[0126] A duplex between polymer 2 and oligo N1 was formed by contacting8.66×10¹³ copies of oligo N1 with the equivalent amount of positivecharges of polymer 2. The reaction was carried out at room temperaturefor thirty seconds in 2 μL of water. One μl of the duplex mixture wasdiluted in 2 mL of water and put into a quartz cuvette for measurementof the initial fluorescence signal. Thereafter, a triplex was formed at42.5° C. by adding and mixing 8.66×10¹³ copies of heat treated (2minutes at 95° C.) NAC1 messenger RNA. Fluorescence of the triplex wasmeasured. A similar duplex and triplex was also made using the DNAoligonucleotide Y2, which has no significant homology with the sequenceof the NAC1 messenger RNA.

[0127] The fluorescence was significantly higher for the N1/polymer2/NAC1 triplex than for the Y2/polymer 2/NAC1 triplex, thereby showingthe ability of polymer 2 to distinguish between specific and nonspecifichybridization of 20 mers DNA oligonucleotides with messenger RNA.

[0128] In conclusion, a novel methodology has been developed that allowsthe detection of nucleic acids by simple optical and electrochemicalmeans. This rapid, selective, and versatile method does not require anychemical reaction on the probes or the analytes, and is based ondifferent electrostatic interactions and conformational structuralchanges between cationic poly(3-alkoxy-4-methylthiophene) derivativesand single-stranded oligonucleotides or double-stranded (hybridized)nucleic acid fragments. The present polymer-based technology is simpleand specific and provides a flexible platform for the rapid detection ofnucleic acids.

[0129] The polythiophenes are thermostable and autoclavable, henceallowing for a wide range of applications.

[0130] The terms and descriptions used herein are preferred embodimentsset forth by way of illustration only, and are not intended aslimitations on the many variations which those of skill in the art willrecognize to be possible in practicing the present invention, as definedin the following claims.

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1. A thiophene monomer having the following general formula:

wherein: a) “m” is an integer ranging from 2 to 3; b) R* is a quaternaryammonium; c) Y is an oxygen atom or a methylene; and d) R¹ is a methylgroup or a hydrogen atom.
 2. A monomer as defined in claim 1, wherein:a) “m”=3; b) R* is +NEt₃; c) Y is an oxygen atom; and d) R¹ is a methylgroup.
 3. A monomer as defined in claim 1, wherein: a) “m” is =2; b) R*is

c) Y is an oxygen atom; and d) R¹ is a methyl group.
 4. A monomer asdefined in claim 1, wherein: a) “m” is =2; b) R* is

c) Y is an oxygen atom; and d) R¹ is a methyl group.
 5. A monomer asdefined in claim 1, wherein: a) “m” is =2; b) R* is

c) Y is a methylene group; and d) R¹ is a hydrogen atom.
 6. The monomeras defined in claim 2, having the formula:


7. The monomer as defined in claim 3, having the formula:


8. The monomer as defined in claim 4, having the formula:


9. The monomer as defined in claim 5, having the formula:


10. A polymer comprising a plurality of monomeric units as defined inany one of claims 1 to
 9. 11. A process for the preparation of a monomeras defined in claim 2, comprising: a) reacting a molecule of formula 1:

with a reagent of formula 2:

in the presence of a copper halide, said reaction resulting in theformation of a first reaction mixture comprising a compound of formula3:

b) isolating said compound of formula 3 from said first reactionmixture; c) reacting said compound of formula 3 with ethylbromide,resulting in the formation of a second reaction mixture comprising saidmonomer; and d) recovering said monomer from said second reactionmixture.
 12. The process of claim 11, wherein said copper halide is Cul.13. A process for the preparation of a monomer as defined in claim 3,comprising: a) reacting a compound of formula 4:

with 1-methylimidazole, resulting in the formation of a reactionmixture; and b) recovering said monomer from said reaction mixture. 14.A process for the preparation of a monomer as defined in claim 4,comprising: a) reacting a compound of formula 4:

with 1,2-dimethylimidazole, resulting in the formation of a reactionmixture; and b) recovering said monomer from said reaction mixture. 15.A process for the preparation of a monomer as defined in claim 5,comprising: a) reacting a compound of formula 6:

with 1-methylimidazole, resulting in the formation of a reactionmixture; and b) recovering said monomer from said reaction mixture. 16.A process for polymerizing the monomer defined in any one of claims 1 to9, comprising reacting said monomer in the presence of an oxidizingagent, resulting in the formation of a polymer of the following generalformula:

wherein: a) “m” is an integer ranging for 2 to 3; b) “n” is an integerranging from 3 to 100; c) R* is a quaternary ammonium; d) Y is an oxygenatom or a methylene; and e) R¹ is a methyl group or a hydrogen atom. 17.The process of claim 16, wherein said oxidizing agent is FeCl₃ orK₂S₂O₈.
 18. The process of claim 16, wherein when R¹ is a methyl group,said polymer is regio-regular and water soluble.
 19. The process ofclaim 18, wherein said polymer is thermostable.
 20. A polymer obtainedfrom the process of anyone of claims 16 to
 18. 21. A method fordetecting the presence of a negatively charged polymer, comprising thesteps of: a) contacting a complementary target to said polymer with apolymer as defined in claim 20 to form a duplex; b) contacting saidduplex with said negatively charged polymer; and c) detecting a changein electronic charge, fluorescence or color as an indication of thepresence of said negatively charged polymer.
 22. A method as defined inclaim 21, wherein said negatively charged polymer is selected from thegroup consisting of: acidic proteins, glycosaminoglycans, hyaluronans,heparin, chromatographic substrates, culture substrates and nucleicacids.
 23. A method as defined in claim 22, wherein said negativelycharged polymer is a nucleic acid.
 24. A method as defined in claim 23,wherein said nucleic acid is DNA.
 25. A method as defined in claim 23,wherein said nucleic acid is RNA.
 26. A method as defined in claim 23,wherein said complementary target is a nucleic acid complementary tosaid negatively charged polymer.
 27. A method as defined in claim 26,wherein said complementary target is a nucleic acid probe and saidnegatively charged polymer is a denatured PCR amplicon.
 28. A method fordiscriminating a first nucleic acid from a second nucleic acid, saidsecond nucleic acid differing from said first nucleic acid by at leastone nucleotide, comprising the steps of: e) contacting a complementarytarget to said first nucleic acid with a polymer as defined in claim 20to form a duplex; f) contacting said duplex with said first nucleicacid, resulting in specific hybridization; g) contacting said duplexwith said second nucleic acid, resulting in non-specific hybridization;and h) detecting a change in electronic charge, fluorescence or color.29. A method as defined in claim 28, wherein said change in electroniccharge, fluorescence or color is more pronounced with said specifichybridization.
 30. A method as defined in claim 29, wherein saidcomplementary target is a nucleic acid complementary to said firstnucleic acid.
 31. A method as defined in claim 30, wherein said firstnucleic acid and said second nucleic acid are DNA.
 32. A method asdefined in claim 30, wherein said first nucleic acid and said secondnucleic acid are RNA.
 33. A method as defined in claim 30, wherein saidcomplementary target is a nucleic acid probe and said first nucleic acidand said second nucleic acid are denatured PCR amplicons.
 34. Use of apositively charged polymer comprising a repeating thiophene moiety fordetecting the presence of a negatively charged polymer.
 35. A use asdefined in claim 34, wherein said positively charged polymer is reactedwith a complementary target to said negatively charged polymer.
 36. Ause as defined in claim 35, wherein said positively charged polymer isionically linked to said complementary target.
 37. A use as defined inclaim 34, wherein a complementary target to said negatively chargedpolymer is linked to a solid support, said negatively charged polymer isthen reacted with said complementary target which results in its captureby said solid support resulting in the formation of a complex and thensaid positively charged polymer is reacted with said complex of saidcomplementary target with said negatively charged polymer.
 38. A use asdefined in claim 34, wherein a complementary target to said negativelycharged polymer is linked to a solid support, said positively chargedpolymer is then reacted with said complementary target resulting in itscapture by said solid support forming a complex, and then reacting saidnegatively charged polymer with said complex of said complementarytarget and said positively charged polymer.
 39. A use as defined inclaim 37 or 38, wherein said solid support is selected from the groupconsisting of an electrode, an optical fiber, a glass slide and glassbeads.
 40. A use as defined in any one of claims 37-39 for purifyingnegatively charged polymers.
 41. A use as defined in claim 40, whereinsaid negatively charged polymers are nucleic acids.